The main objective of this project is to dissect the underlying mechanisms of the transcriptional network linking the survival pathway to the differentiation program. This work focuses on the transcriptional dynamics controlled by the neuronal-specific basic Helix-Loop-Helix (bHLH) transcription factor, Nex1/Math-2. It is uniquely expressed in the future language during the left-right brain development as well as other areas associated with cognitive functions. Our studies show that Nex1 is a key regulator of the neuronal differentiation program, as its expression is critical to the execution of the NGF-induced differentiation pathway in PC12 cells. Its overexpression induces spontaneous neuritogenesis, accelerated NGF-induced differentiation, and neurite regeneration. Most importantly, our studies reveal the first evidence that Nex1 displays neuro-protective properties since it: 1) prevents apoptosis of PC12-Nex1 cells upon trophic factor deprivation;and 2) concomitantly modulates the expression of key anti-apoptotic and cell cycle regulators. Accordingly, we hypothesize that Nex1 promotes survival by orchestrating several distinct but interconnected programs, linking neuronal differentiation to cell cycle withdrawal and the anti-apoptotic pathway. To test our overarching hypothesis and elucidate the transcriptional network of Nex1, we plan to execute the three following aims: Aim I will investigate the role of Nex1 in the E2F-Rb pathway, a critical component of the NGF pathway, linking cell cycle arrest to survival. The functional participation of E2F-Rb members will be tested using siRNA-based gene knockdown combined with cell cycle and apoptosis-based flow cytometry assays. Aim II will elucidate the specific Nex1-mediated transcriptional network by performing genomic analyses restricted to the cell cycle, apoptosis, and neuronal-related pathways. The functional roles of Nex1-regulated genes will be assessed by siRNA-based silencing and functional flow cytometry-based cell cycle/apoptosis assays, at different phases of Nex1-mediated survival. Furthermore, direct target genes of Nex1 will be identified. The combined results obtained in the PC12 cell system from Aim I and II will be validated in cortical and cerebellar granule neurons cultures with their respective cell death paradigms. Aim III will dissect the transcriptional regulation of the Bcl-w gene, which we have identified and characterized as being a Nex1 target gene. The identity and functions of the critical regulatory elements will be analyzed during neuronal differentiation and survival, using DNasel footprinting, EMSA, and luciferase assays. Accordingly, Aim III addresses the NIH ENCODE initiative to unravel critical functional elements in the human genome, and to link SNPs mapping in key regulatory elements with neuronal-related diseases. Ultimately, the combined differentiation and neuro-protective properties of Nex1 may have broad implications for the design of novel therapeutic approaches to treat neurodegenerative diseases and CNS injuries.